This invention relates to delivering diagnostic or biologically active compounds to targeted cells and controlled release of these compounds.
A reoccurring problem in medicine is that due to the lack of specificity of the agents used for treatment of a disease, the patient is often the recipient of a new set of maladies from the therapy. This scenario is especially common in the treatment of various forms of cancer where a therapeutic agent will exert its toxic affect on nontargeted normal tissues, in addition to cancerous cells.
To circumvent the nonspecificity of a therapeutic agent, the agent can be coupled to a carrier that possesses some degree of specificity for a particular cell. For example, carrier molecules such as liposomes, proteins and antibodies have been used in conjunction with a broad spectrum of diagnostic and therapeutic agents.
The development of monoclonal antibodies (MAbs), which have a specific recognition and affinity for certain selected antigens, has led to the delivery of diagnostic therapeutic agents to selected cells via immunoconjugates. "Immunoconjugates" are covalently bonded hybrid molecules composed of a target directing moiety (recognition portion), such as an antibody molecule, and an effector molecule, such as a toxin, drug, biological response modifier, or a radioisotope.
Although the MAb carrier system enhances delivery of the therapeutic agent to specific targeted cells, other problems remain. One problematic area includes situations where the agent is only active, or at least more potent, when free from the MAb carrier. Another problematic area is that although MAb are designed to be specific for a particular antigen, some sorption or binding to nontargeted cells will occur resulting in destruction of normal cells. The latter problem occurs, for example, when the immunoconjugate contain a radionuclide. The radioactivity has a tendency to localize nonspecifically in the kidney, potentially exerting undesirable renal toxicity and thereby lowering the therapeutic index of the immunoconjugate. To overcome these noted problems, linkers, which act as a bridge to join the therapeutic agent and and target directing moiety, have been developed which have a cleavable bond to release the therapeutic agent. In the latter situation, where there is nonspecific binding to cells, the agent can be cleaved from the carrier and the agent rapidly cleared from the body.
A number of different cleavable linker groups have been previously described. The mechanism for release of an agent from these linker groups include cleavage by reduction of a disulfide bond, by irradiation of a photolabile bond, by hydrolysis of derivatized amino acid side chain, by serum complement-mediated hydrolysis and acid-catalyzed hydrolysis. [See for example, U.S. Pat. No. 5,053,503, PCT Publication WO 86/00527, PCT Publication WO 90/03188, PCT Publication WO 90/03401, PCT Publication WO 90/14844, European Patent Application 0 253 220, and European Patent Application 0 318 948. Some of these mechanisms are susceptible to release of the agent prior to having reached the specific cell, tissue or organ.
U.S. Pat. No. 4,631,190 and Shen et al. Biochem. Biophys. Res. Commun., 102:1048-1054 (1981) disclose a drug attached to the linker through an acidic bonding group, such as carboxyl, that is in a cis configuration with another acidic group, and a targeting moiety which is attached in a cis configuration with another acidic group. In particular, the antitumor antibiotic daunomycin was linked to poly(D-lysine) using cis-aconitic anhydride and a carbodimide coupling agent. In conjugated form, the nitrogen group of daunomycin exists as an amide, the amide compound is biologically inert. When the conjugate is carried into the cell, the acidic lysosomal environment facilitates hydrolysis of the amide bond to generate free daunomycin.
When using essentially the same method to attach a monoclonal antibody specific for cancer cells to daunomycin with cis-aconitic anhydride, the immunoconjugate was found to be selectively carried into tumor cells where hydrolysis took place to generate free daunomycin. The presence of free daunomycin led to tumor regression in animal tests. [See, for example, Benton et al., in Anthracycline Antibiotics, H. S. El Khadem, Ed., Academic Press, New York pp. 1-57 (1982); Oki, Stud. Biophys., 104:169-200 (1984); Kumar et al., Can. J. Chem., 62:2585-2591 (1984): and Cassinelli, J. Antibiot., 38:856-867 (1984)]. (Control experiments with antibody alone, drug alone, or a mixture of antibody and free drug (unconjugated) resulted in an inferior response.)
Blattler et al., in U.S. Pat. No. 4,569,789, describe a drug delivery system which is formed by reaction of an active substance with a maleic anhydride moiety. The active substance is released upon cleavage of the amide bond. The patent discloses that at pH 5, 15 percent is cleaved after five hours and after five hours at pH 4, less than 50 percent is cleaved.
Blattler et al., in U.S. Pat. No. 4,764,368 disclose heterobifunctional reagents, developed from cyclohexene-maleamic acid, that are cleavable under mildly acidic condition. The heterobifunctional reagents were used for linking the drug gelonin to antibodies, which provides for intracellular release of the drug at the targeted cells. No data was presented for cleavage of the drug from the conjugate.
Thus there remains a need for a carrier agent conjugate which releases the agent by cleavage under mild conditions. Furthermore, it would be advantageous to provide a complex that does not readily dissociate outside the targeted cell, exhibits rapid whole body clearance except from the desired tissue, and conjugates readily with an effector molecule.